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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-11-30 till 2009-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: "Kanpoan No. 287 -- Environment Protection Agency" "Eisei No. 127 -- Ministry of Health &Welfarew "Heisei 09110131 Kikyoku No. 2 -- Ministry of International Trade & Industry"
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): FAT 40849/A TE
- Substance type: reactive dyestuff for cellulose fibers
- Physical state: blue powder
- Analytical purity: 76.9% of all colored components
- Lot/batch No.: TZ 5463 / BOP 03-09
- Expiration date of the lot/batch: 2014-06-30
- Storage condition of test material: At room temperature at about 20 °C

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 Lg/plate
Vehicle:
deionised water
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 2-aminoanthracene, 4-nitro-o-phenylene-diamine, methyl methane sulfonate
Details on test system and conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 1 hour
- Exposure duration: approx 72h



NUMBER OF REPLICATIONS: 3 plates



DETERMINATION OF CYTOTOXICITY
- Method:reduction in the number of revertants (below the indication
factor of 0.5)
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks:
(not relevant)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar from 1000 Lg/plate up to 5000 Lg/plate in both experiments. No precipitation was observed on the incubated agar plates. The undissolved particles had no influence on the data recording


RANGE-FINDING/SCREENING STUDIES:The pre-experiment is reported as experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA:the spontaneous reversion rates in the negative and solvent control are in the range of
our historical data

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test item showed normal background growth up to 5000 Lg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication
factor of 0.5), occurred in almost all strains the with and without metabolic activation. A
minor reduction in the number of revertants (below the indication factor of 0.5), occurred in
strain TA 1537 and TA 98.

Any other information on results incl. tables

Summary of Results Pre-Experiment and Experiment I

 

Metabolic Activation

Test Group

Dose Level (per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

Deionised water

 

12 ± 2

15 ± 0

25 ± 2

130 ± 16

44 ± 9

Untreated

 

11 ± 2

15 ± 1

25 ± 6

127 ± 17

44 ± 12

FAT 40849/A

TE

     3μg

11 ± 2

 8 ± 3

28 ± 4

127 ± 1

50 ± 4

   10μg

12 ± 2

 9 ± 4

23 ± 2

121 ± 12

48 ± 9

   33μg

13 ± 1

12 ± 0

22 ± 6

121 ± 6

46 ± 2

 100μg

 9 ± 1U M

 9 ± 2

24 ± 9

134 ± 1

50 ± 1

 333μg

 7 ± 2U M

 6 ± 3

26 ± 5

116 ± 18

50 ± 2

1000μg

 9 ± 2U M

 5 ± 2

29 ± 1

109 ± 2

51 ± 1

2500μg

12 ± 3D M U

 3 ± 3D M

16 ±D M

113 ± 8D M

43 ± 4D M

5000μg

 8 ± 3D M U

 6 ± 2D M

17 ± 4D M

114 ± 8D M

47 ± 3D M

NaN3

   10μg

1884 ± 28

 

 

2059 ± 181

 

4-NOPD

   10μg

 

 

220 ± 2

 

 

4-NOPD

   50μg

 

339 ± 2

 

 

 

MMS

  3.0μL

 

 

 

 

1161 ± 26

With Activation

Deionised water

 

14 ± 1

12 ± 2

34 ± 7

140 ± 7

60 ± 2

Untreated

 

15 ± 1

15 ± 5

38 ± 5

150 ± 10

63 ± 14

FAT 40849/A TE

      3μg

16 ± 4U M

13 ± 1

35 ± 6

142 ± 18

62 ± 6

   10μg

11 ± 3U M

10 ± 2

35 ± 6

151 ± 18

58 ± 7

   33μg

13 ± 4U M

14 ± 2

29 ± 4

130 ± 10

56 ± 8

 100μg

11 ± 1U M

 9 ± 4

36 ± 2

153 ± 16

63 ± 9

 333μg 

14 ± 3U M

 8 ± 1

33 ± 7

139 ± 11

68 ± 9

1000μg

14 ± 1U M

 7 ± 0

18 ± 1

141 ± 2

69 ± 2

2500μg

10 ± 1D M U

 5 ± 1D M

18 ± 2D M

116 ± 4D M

43 ± 4D M

5000μg

10 ± 4D M U

 5 ± 2D M

20 ± 3D M

102 ± 11D M

47 ± 3D M

2-AA

  2.5μg

383 ± 34 

381 ± 75

1513 ± 175

2476 ± 79

 

2-AA

 10.0μg

 

 

 

 

234 ± 20

 

 

 

 

Summary of Results Experiment II

 

Metabolic Activation

Test Group

Dose Level (per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

Deionised water

 

18 ± 6

13 ± 1

24 ± 4

130 ± 10

52 ± 7

Untreated

 

19 ± 4

11 ± 4

25 ± 2

130 ± 11

48 ± 7

FAT 40849/A

TE

        3μg

15 ± 5

12 ± 3

32 ± 3

122 ± 4

52 ± 7

     10μg

17 ± 4

10 ± 3

29 ± 7

125 ± 9

44 ± 3

     33μg

17 ± 1

15 ± 6

32 ± 4

129 ± 7

50 ± 11

   100μg

14 ± 2

10 ± 3

33 ± 6

136 ± 3

60 ± 6

   333μg

15 ± 3

10 ± 3

31 ± 6

132 ± 4

47 ± 7

 1000μg

11 ± 4

9 ± 1

24 ± 4

118 ± 23

38 ± 11

 2500μg

14 ± 4D M

6 ± 1D M

19 ± 3D M

133 ± 8D M

49 ± 6D M

 5000μg

10 ± 4D M

7 ± 4D M

20 ± 7D M

156 ± 10D M

60 ± 6D M

NaN3

     10μg

1925 ± 76

 

 

2118 ± 39

 

4-NOPD

     10μg

 

 

382 ± 68

 

 

4-NOPD

     50μg

 

113 ± 10

 

 

 

MMS

    3.0μL

 

 

 

 

676 ± 57

With Activation

Deionised water

 

18 ± 5

16 ± 9

37 ± 4

156 ± 24

67 ± 14

Untreated

 

16 ± 4

17 ± 9

36 ± 7

155 ± 7

58 ± 8

FAT 40849/A

TE

      3μg

21 ± 8

17 ± 4

38 ± 6

146 ± 15

57 ± 9

    10μg

18 ± 8

16 ± 1

41 ± 1

159 ± 9

56 ± 14

    33μg

18 ± 4

14 ± 2

32 ± 8

154 ± 8

64 ± 9

   100μg

22 ± 7

16 ± 5

33 ± 8

160 ± 17

59 ± 10

   333μg

14 ± 2

19 ± 5

31 ± 5

162 ± 5

78 ± 11

 1000μg

14 ± 6

11 ± 3

20 ± 2

136 ± 10

44 ± 1

 2500μg

14 ± 4D M

 8 ± 1D M

17 ± 2D M

134 ± 7D M

60 ± 9D M

 5000μg

14 ± 6D M

 6 ± 2D M

16 ± 3D M

150 ± 3D M

74 ± 4D M

2-AA

   2.5μg

346 ± 10

234 ± 28

2089 ± 162

2274 ± 50

 

2-AA

 10.0μg

 

 

 

 

399 ± 21

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

NaN3 sodium azide

D Densely coloured plate

2-AA 2-aminoanthracene

M Manual count

4-NOPD 4-nitro-o-phenylene-diamine

U Air bubbles

MMS methyl methane sulfonate

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, FAT 40849/A TE is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of FAT 40849/A TE to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 Lg/plate

The plates incubated with the test item showed normal background growth up to 5000 Lg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in almost all strains the with and without metabolic activation. A minor reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA 1537 and TA 98.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40849/A TE at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.